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Mechanical cues play important roles in controlling cell function and have implications fo1' generating functional tissues for clinical use. This thesis investigates the potential of using magnetic particle (MNP) mediated mechanical stimulation techniques through Magnetic Force Bioreactor (MFB) to alternatively activate mechanical sensitive cell membrane receptors for the tissue engineering applications and the study of mechanotransduction. In this study, broad cell types including primary human mesenchymal stem cells (hMSCs), transformed mouse embryonic stem cells (mESCs) and HEK293 cells have been used and two different strategies for improving selective targeting cell membrane receptors, in that antibodies or peptides are employed as functionalizing biomolecules to facilitate various cell membrane receptors targeting, have been utilized. The long term application of mechanical stimulation in the osteogenic induction of hMSCs and mESCs mediated by MNPs has been undertaken. The cellular response to MNP mediated stimulation has becn examined by immunofluorescence, ELISA, qRT-PCR, histology, FTIR and uCT.

Abstract Muscle satellite cells are a population of undifferentiated mononuclear myogenic progenitor cells. The aim of this study was to explore the developmental origin of satellite cells in Xenopus laevis embryos and identify the function of Xenopus pax7, a reliable marker of Xenopus satellite cells, in satellite cell development and muscle regeneration.

Summary Tissue engineering and regenerative medicine strategies until now have mostly relied on static culture using chemical stimulation to induce cell differentiation. However, these strategies neglect the dynamic environment in which cells reside in the body where they are surrounded by a chemically and physically well-defined threedimensional (3D) topography. Not only does this environment control cellular differentiation, but its structure also determines the mechanical function of that tissue. Alongside physical cues, external mechanical forces play an essential role in the homeostasis of many tissues, particularly bone. In order to develop tissue engineered constructs that are suitable for implantation, it may be important to incorporate these essential cues into pre-culture methods and in order to do this, a better understanding of the cellular responses is required. The main aim of this research was to understand how physical and mechanical cues affect cell behaviour, differentiation and matrix production, with particular emphasis on osteogenesis and collagen organisation. In order to achieve this, electrospun scaffolds were fabricated with controllable fibre orientation for studies involving fibroblast matrix organisation, and the affect on the differentiation of osteoprogenitor cells. Short bouts of tensile loading were conducted using a previously established bioreactor model for conditioning collagen-producing cells. A simple rocking platform method for subjecting cells to fluid-flow was also investigated for its potential to enhance osteogenesis and collagen organisation. This system was further used to study the role of the primary cilium for the mechanotransduction of bone cells. The overall goal was to understand how to manipulate cell differentiation and matrix production in order to develop a more suitable construct with correct tissue structure in a rapid manner. Monitoring of the major structural matrix protein collagen was achieved using the minimally-invasive technique of second harmonic generation, which was optimised. Electrospun scaffolds with a random architecture caused cells to deposit matrix in a similar random manner, however highly aligned scaffolds caused deposited collagen to orientate in the fibre direction giving superior tensile properties. Further to this, random fibres appeared to be more favourable for the differentiation of osteoprogenitor cells than highly aligned substrates. 9 Short bouts of tensile stimulation of collagen producing cells on 3D substrates caused an increase in collagen deposition. Another stimulation method, a simple rocking platform, created oscillatory fluid shear stress (FSS) suitable for stimulation of osteogenic cells and enhanced collagen organisation. Further to this, human dermal fibroblasts could be induced to form a mineralised matrix when cultured in osteogenic media, which was further enhanced with FSS. It was also demonstrated that this simple rocking system could be used to test a wide variety of loading parameters. Finally, rocking was used to examine the role of the primary cilium in the load-induced mineral deposition response of bone cells. When mature bone cells were subjected to FSS, primary cilia shortened in length and removal of primary cilia resulted in loss of the load-induced matrix response suggesting that primary cilia are mechanosensors in bone cells.

Even relatively simple microorganisms, which have been extensively studied, are hosts to a complex network of interconnected processes occurring on diverse time scales. The multilevel nature of the regulatory network of cells and the interactions occurring at the intra cellular level further augment this complexity (Yokobayashi et al., 2003). Attempts to wholly model the function of even a single cell are currently non trivial, if not impossible, as the amount of delicate intracellular measurements required to validate such a model is exhaustive both in terms of labour and cost. Uncertainties introduced both on the parameter identifiability and on the mechanistic level further complicate this task. The large number of biological data generated with the advancement of a variety of high-throughput experimental technologies demand for the development of comprehensive mathematical model building methods able to capture the complex phenomena occurring within a cell (Covert et al., 2001). Borrowing the fundamental research principles from the Process Systems Engineering paradigm, mathematical modelling of biological systems can provide a systematic means to quantitatively study the characteristics of the multilevel interactions that occur in cell culture. In the present thesis, an integrated modelling framework is established that can ensure the seamless interaction of experimental biology with the development of quantitative mathematical descriptions of biological systems. The use of model-based techniques can facilitate the reduction of unnecessary experimentation and reduce labour and operating costs by identifying the most informative experiments and providing strategies to optimise and automate the bio-process at hand. Paving the way towards a ‘closed-loop’ approach for bio-process automation (Kiparissides et al., 2011), the work herein presents a biological model development framework following a step by step approach, highlighting challenges and “real life” problems associated with each stage of model development. By organising available information in a systematic way, unnecessary experimentation is avoided and models with a priori objectives can be established to guide the in vivo process through the in silico representation. The proposed methodology combines macroscopic and subcellular model development, parameter estimation, global sensitivity analysis, model based design of experiments and selection of optimal feeding policies via dynamic optimisation methods in a fromalised structure. The combined mathematical and experimental framework for the control and optimisation of mammalian cell culture systems, presented herein, is experimentally validated via the succesfull model based optimisation of antibody secreting GS-NS0 cell cultures.

Conventional methods of combining mammalian cells and synthetic polymers for tissue engineering applications are frequently problematic. This is due to the incompatibility between the sensitive cell component and the harsh polymer processing environments required to form the desired porous scaffold e. g. high temperatures and organic solvents. This results in the necessity for an often inefficient and time consuming two step scaffold seeding process, whereby mammalian cells are added to a pre-fabricated polymer scaffold. High pressure or supercritical CO2 (scCO2) processing is a method of fabricating porous polymer scaffolds at ambient temperatures and without using organic solvents. When pressurised, CO2 becomes highly soluble in a variety of amorphous polymers such as poly(DL-lactic acid) (PDLLA) to produce a high viscosity liquid. Subsequent decompression causes the formation of gas bubbles that become permanent as the polymer vitrifies. Based upon technology at the University of Nottingham, we hypothesised that mammalian cells could be incorporated into poly(DL-lactic acid) (PDLLA) scaffolds using a single step scCO2 process. This would not only make the process more rapid, but it would remove the inefficient scaffold seeding step required in most cell based tissue engineering strategies. Mammalian cells were subject to a range of high pressure CO2 and N2 processing conditions and assessed for cell survival. It was discovered that primary hepatocytes, meniscal fibrochondrocytes, myoblastic C2C12s and 3T3 fibroblasts could survive after exposure to both high pressure gases on a time and pressure dependent basis. Cells exposed to scCO2 for one minute were then assessed for both gene and enzyme function.Using a microarray, it was found that only eight genes (out of 9000) in murine C2C12 cells were significantly down-regulated when compared to untreated cells. Continued cell function was confirmed by measuring BMP-2 induced alkaline phosphatase activity as a measure of osteogenic differentiation in myoblastic C2C12 cells. Alkaline phosphatise activity was indistinguishable between untreated cells and cells exposed to scCO2 for one minute. Additional enzyme and receptor function was confirmed by measuring cytochrome P450 activity in primary hepatocytes after one minute of scCO2 processing. In the second half of the study, these short processing times were found to be sufficient to plasticise and foam porous PDLLA scaffolds. Therefore, cells were incorporated into the biodegradable PDLLA foams by pre-mixing the cell suspension with the polymer powder and exposing to scCO2. Subsequent decompression caused the polymer to foam with the cells trapped within the porous structure. Despite the presence of the plasticised PDLLA, cell survival was confirmed by both an Alamar B1ueTM assay and LIVE/DEADTM staining. Osteogenic differentiation on the scaffolds was confirmed by a stain and assay for BMP-2 induced alkaline phosphatase activity. Finally, a second generation processing piece of processing apparatus was designed that permitted mammalian cells to be passed into a pressurised vessel containing preplasticised PDLLA using a novel high-pressure CO2 injection system. This was made possible by constant optimisation of the high pressure apparatus and the introduction of a cell delivery valve. When injected at high pressures cell survival was found to be reduced when compared with previous experiments although this was likely to be due to the additional mechanical trauma caused by the injection process. Despite this, the live cell population was shown to retain its osteogenic differentiation capacity when induced with BMP-2. With further optimisation of the delivery method, cells may survive this process in sufficient numbers to suggest that it could be used as a method of seeding tissue engineering scaffolds in the future. This development could remove the limitations place on polymer processing time by the finite survival period of the cells, permitting tuning of the scaffold structure to suit the application. In summary, this study has demonstrated that mammalian cells can be incorporated into biodegradable PDLLA scaffolds using a rapid, one-step scCO2 process without the use of toxic solvents or elevated temperatures. Furthermore, the development of the high pressure injection system could allow cells to be incorporated during the fabrication step, removing the restrictions on polymer processing. This technique could be used for the rapid production of tissue cell loaded engineering scaffolds and other associated biotechnological applications where cells and synthetic polymers are combined, such as cell therapy and recombinant protein production.

Pre-cellularised scaffolds are limited in volume due to the constraints of the time delay required for angiogenic cells ingrowth forming a vascular network and allowing for delivery of nutrients and waste exchange. Channels have the potential to improve the time taken for cellular penetration. The effectiveness of channels in improving angiogenic cells penetration was assessed in vitro and in vivo in porous 3-D collagen scaffolds. Initial studies conducted in vitro demonstrated that the scaffolds supported angiogenic cells ingrowth in culture and the channels improved the depth of penetration of cells into the scaffold. The cells reside mainly around the channels and migrate along the channels. In vivo, channels increased cell migration into the scaffolds and in particular angiogenic cells resulting in a clear branched vascular network of micro vessels in the channelled samples which was not apparent in the non-channelled samples. This correlated well with macrophage invasion into scaffolds since angiogenesis in vivo is usually accompanied by infiltration of macrophages which participate in organization of angiogenesis, and in regulation of tissue regeneration. Thus, macrophage-mediated biodegradation of collagen scaffolds in vitro was also assessed. Furthermore, pre-seeding channelled collagen scaffolds with endothelial cells implantation has potential of speeding up vascularisation of scaffolds compared to human bone marrow stromal cells.

Στην ιστοτεχνολογία (tissue engineering – TE), η ικανότητα των ικριωμάτων (scaffolds) να διατηρούν συμβατή μηχανική και βιολογική συμπεριφορά με τους περιβάλλοντες ιστούς και όργανα, αλλά και να διευκολύνουν την προσκόλληση κυττάρων του ξενιστή στην επιφάνεια και την τρισδιάστατη δομή τους κρίνεται ως υψίστης σημασίας για την επιθυμητή εκδήλωση αναγεννητικής αντίδρασης των κυττάρων in vivo. Ο σκοπός της παρούσας διδακτορικής διατριβής είναι η δημιουργία και ο χαρακτηρισμός ακυτταροποιημένης εξωκυττάριας μήτρας από μαλακούς ιστούς ζωικής προέλευσης, με στόχο τη δημιουργία ικριώματος με επιθυμητές μηχανικές και βιολογικές ιδιότητες για εφαρμογές στην ιστοτεχνολογία. Στην εργασία αυτή επιλέχθηκε ως υλικό της μελέτης ο βόειος περικαρδιακός ιστός, λόγω της ευρείας πολύχρονης χρήσης του ως βιοϋλικό σε μοσχεύματα. Εφαρμόστηκαν δύο διαφορετικά πρωτόκολλα για την ακυτταροποίηση του ιστού, χρησιμοποιώντας στο πρώτο Triton Χ-100, SDS και deoxycholic acid (12 ώρες, 4°C - Triton) και στο δεύτερο Trypsin/EDTA με RNAse/DNAse (48 ώρες, 37°C – Trypsin). Η ιστολογική εξέταση επιβεβαίωσε την ολική αφαίρεση των κυττάρων. Τα αποτελέσματα των εμβιομηχανικών δοκιμών δεν έδειξαν στατιστικά σημαντική διαφορά μεταξύ των μηχανικών ιδιοτήτων των μη κατεργασμένων ιστών και των ακυτταροποιημένων περικαρδιακών ιστών με Triton στην οριζόντια και κάθετη διεύθυνση προς τον οβελιαίο άξονα της καρδιάς σε αντίθεση με τη μείωση του χαμηλού μέτρου ελαστικότητας (φάση ελαστίνης) και του υψηλού μέτρου ελαστικότητας (φάση κολλαγόνου) μετά την κατεργασία των υλικών με Trypsin, και στις δύο κατευθύνσεις. Η βιοχημική ανάλυση επαλήθευσε την άμεση σχέση μεταξύ των μηχανικών βισκοελαστικών ιδιοτήτων των μαλακών ιστών με τα συστατικά (GAGs και κολλαγόνο) του ιστού και την εσωτερική διαμόρφωση τους. Τα αποτελέσματα των μη επεξεργασμένων και των επεξεργασμένων ιστών με τα παραπάνω διαλύματα, έδειξαν ότι το διάλυμα του Triton προκαλεί ήπια κατεργασία, με ελαχιστοποίηση των δομικών μεταβολών της εξωκυττάριας μήτρας, όπως η σταθερή σύσταση των γλυκοζαμινογλυκανών (GAGs) και η περιεκτικότητα των ιστών σε κολλαγόνο. Αυτό δεν επιτεύχθηκε με την χρήση διαλύματος Trypsin, όπου παρατηρήθηκε σημαντική μείωση των GAGs, τόσο στη συγκέντρωση της θειϊκής χονδροϊτίνης / δερματάνης όσο και στο υαλουρονικό. Για την αξιολόγηση της κυτταροσυμβατότητας αορτικά βόεια ενδοθηλιακά κύτταρα καλλιεργήθηκαν στα ακυτταροποιημένα υλικά. Η επιθηλιοποίησή τους επετεύχθη από 24 ώρες μέχρι και 4 μέρες. Προσδιορίστηκε επίσης η δύναμη προσκόλλησης των κυττάρων μετά από 4 μέρες καλλιέργειας στους ακυτταροποιημένους περικαρδιακούς ιστούς με την εφαρμογή διατμητικών τάσεων μέσω ροϊκού πεδίου, με χρήση κατάλληλης μηχανής περιστροφής των δειγμάτων σε ακινητοποιημένο υγρό. Τα αποτελέσματα έδειξαν εξάπλωση και πολλαπλασιασμό των κυττάρων στην επιφάνεια των βιοϋλικών, ενώ παρατηρήθηκε καλή συγκέντρωση κυττάρων (> 60%) στην κλίμακα των φυσιολογικών διατμητικών τάσεων. Συμπερασματικά, η ακυτταροποίηση του βόειου περικαρδιακού ιστού για μεγάλη χρονική διάρκεια στους 37°C (Trypsin) φάνηκε να μεταβάλλει την εμβιομηχανική συμπεριφορά και τη δομική ακεραιότητα του ιστού, η οποία, αντίθετα, διατηρείται σε φυσιολογική κατάσταση μετά από κατεργασία σε χαμηλή θερμοκρασία και σε σύντομο χρόνο (Triton). Επιπλέον, και τα δύο πρωτόκολλα είχαν ως αποτέλεσμα τη δημιουργία βιοϋλικού συμβατού με τα ενδοθηλιακά κύτταρα κατά την επαφή τους με την επιφάνειά τους. / In TE scaffolding, the ability of scaffolds to preserve proper mechanical and biological function and compatibility with the surrounding tissues and organs, as well to enhance host cells to adhere with scaffold material in their surface and 3D structure is of paramount importance for potential regenerative cell response in vivo. The aim of the present thesis is to produce and characterise a decellularized extracellular matrix derived from animal soft tissues, scoping to a scaffold capable of exhibiting the mechanical and biological properties desired for tissue engineering (TE) applications. For this work bovine pericardial tissue was selected, due to its broad use as a biomaterial in different implant technologies for decades. Two different protocols for the decellularization of bovine pericardial tissue were developed incorporating Triton® X–100, SDS and deoxycholic acid (12 h, 4°C) in solution 1 (Triton) and trypsin/EDTA with RNAse/DNase at 37°C for 48 h in solution 2 (Trypsin). Histological analysis confirmed total absence of cells after both treatments. The results of the biomechanical tests showed no mechanical differences demonstrated between the fresh and decellularized pericardial tissues by Triton solution in both, apex to base and transverse anatomical directions, contrary to a significant decrease of the low elastic modulus (elastin phase) and high elastic modulus (collagen phase) demonstrated after trypsin solution treatment in both directions. Biochemical analysis verified the direct relationship between mechanical viscoelastic properties of soft tissues with the constituent tissue components (GAGs and collagen content) and their internal arrangement. The comparison of the results from untreated and that from treated tissues suggested that the Triton decellularization method seemed to be a very mild treatment, as the glycozaminoglycans (GAGs) composition remained constant, as well as the collagen content. This was not achieved with the decellularization using Trypsin, where a significant reduction of GAGs, both chondroitin/dermatan sulphate and hyaluronan, was found. Aortic bovine endothelial cell seeding was used for estimation of its biomaterials’ cytocompatibility. Endothelialization achieved after 24hrs to 4 days periods. The adhesion strength of cells, cultured for 4 days on decellularized bovine pericardial tissues, was also determined. By applying a radially increasing shear stress field on rotating material samples within a stationary fluid mesh using a spinning disc device, we determined the shear stress necessary to detach the cells from the sfafolds’ surface. Fine cell spreading and proliferation on biomaterials’ surface and good surface cell density (>60%) at physiological shear stress scale were observed and measured. In conclusion, decellularization of bovine pericardial tissues under long time duration in 37°C (Trypsin) seems to alter its biomechanical behaviour and structural integrity, which, in contrast, was retained under low temperature short duration treatment (Triton). Additionally, both protocols resulted in a cytocompatible biomaterial, regarding its surface interactions with endothelial cells.